It is known in so-called STED fluorescence optical microscopy for a sample which has first of all been excited to fluoresce by means of excitation light to be deexcited again with deexcitation light down to a spatially tightly bounded area before the detection of fluorescence light which is emitted spontaneously from the sample. If this area is the null of an interference pattern of the deexcitation light and the intensity of the deexcitation light is high, it is possible to reduce the spatial dimensions of this area from which the spontaneously emitted fluorescence light can exclusively originate below the diffraction limit for the wavelength of the light being used. This results in a considerable increase in the spatial resolution for imaging of the sample by means of the spontaneously emitted fluorescence light.
The interference pattern of the deexcitation light is frequently produced with the aid of a phase filter or spatial light modulator, by means of which incident planar phase fronts of the deexcitation light are deliberately deformed. For example, the phase fronts can thus be delayed in a central area close to the optical axis, in comparison to the phase fronts in the periphery. During focusing of the deexcitation light, this results in an interference pattern with a null at the focus point, which null, in the direction of the optical axis, lies between two main maxima of the light intensity distribution and on the focal plane within a ring of weak intensity. Another doughnut-shaped interference pattern with a null line along the optical axis through the focus point can be achieved by providing the incident planar wavefronts of the deexcitation light with a phase delay which rises in a helical shape about the optical axis up to a wavelength of the deexcitation light.
In known STED microscopes, the deexcitation light whose phase fronts have been distorted in this way is combined with the excitation light and is directed into an objective lens which focuses both the excitation light and the deexcitation light into the respective sample and also receives fluorescence light coming from the sample (see for example the published German patent application DE 10 2005 020 003 A1). The fluorescence light from the sample can in this case pass back in a known manner essentially on the same path as that over which the excitation light previously passed. The excitation light can thus pass through a pinhole in order to image a point light source in the sample by means of the objective lens, and the fluorescence light from the sample can pass back through a conjugate pinhole, after separation of the excitation light by means of a wavelength-selective beam splitter, thus resulting in a so-called confocal arrangement. One advantage in this case is that all the changes to the optical assembly between the pinhole and the sample affects the excitation light and the fluorescence light in the same way. However, this involves major adjustment effort, in order to coaxially align the beam paths of the excitation light on the one hand and of the deexcitation light on the other hand accurately with respect to one another.
A similar problem occurs in RESOLFT fluorescence optical microscopy, in which the basic state of a fluorescent dye in a sample is first of all depopulated with depopulation light down to a spatially closely confined area before the sample is excited to fluoresce by means of excitation light, in order to detect fluorescence light from the sample. When the spatially closely confined area is the null of an interference pattern of the depopulation light, and the intensity of the depopulation light is high, it is possible to reduce the spatial dimensions of this area, from which the fluorescence light can exclusively originate, below the diffraction limit at the wavelength of the light being used. In this case, this also means a considerable increase in the spatial resolution for imaging of the sample by the fluorescence light. In RESOLFT fluorescence light microscopy, the depopulation light and the excitation light, which may be at the same wavelength, can be combined onto a common optical axis.
In addition to the already mentioned spatial light modulator, the described aberrations of planar wavefronts of the deexcitation light can be effected by phase filters (see for example DE 10 2006 011 556 A1), phase delay elements, e.g. deposited onto a plane-parallel glass plate, or can be produced by a stepped glass plate or a helical glass plate, which is referred to as a phase clock.
Although the use of so-called spatial light modulators is advantageous to the extent that fronts of any desired shape can in theory be generated; it is found, however, that the beam quality is adversely affected by spatial light modulators independently of the applied modulation of the phase shift.
In the case of optical components with phase delay elements and/or stepped or helical thickness, no adverse effect on the deexcitation light passing through can admittedly be expected, apart from the desired variation of the phase fronts, but these components are only suitable for deexcitation light in a narrow wavelength range.
This also applies to a phase filter which applies to linear phase ramps in opposite senses to the light passing through, and which is known from G.-H. Kim, J.-H. Jeon, K.-H. Ko, H.-J. Moon, J.-H. Lee and J.-S. Chang, “Optical vortices produced with a nonspiral phase plate”, Appl. Opt. 36, 8614-8621 (1997).
EP 1 662 296 A1 describes an optical assembly having the features of the preamble of patent claim 1, in which the two light components have different polarization such that the wavefronts of the one light component are deformed by a spatial light modulator, while the wavefronts of the other light component pass through a spatial light modulator without being deformed. The beam paths of the two light components, for example of the excitation light and of the deexcitation light in the case of a STED fluorescence optical microscope, therefore need not be separated. The known adverse effects on the beam quality caused by a spatial light modulator occur in both light components in this case, however.
There is therefore still a requirement for an optical assembly in which, for example, the adjustment effort for the combination of the deexcitation light and of the excitation light in an STED fluorescence optical microscope or the depopulation light and the excitation light in an RESOLFT fluorescence optical microscope can be reduced considerably without any adverse effects on the beam quality.